Antimicrobial peptides derived from ubiquicidine

ABSTRACT

The invention relates to the use of ubiquicidine or optionally modified peptide fragments derived therefrom for the preparation of a drug for the treatment, diagnostics or prophylaxis of infections in humans and animals. A peptide fragment derived from ubiquicidine comprises for instance a preferably continuous series of at least 3, preferably at least 7-13 amino acids from the amino acid sequence of ubiquicidine; KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPN ANS (SEQ ID NO: 1). Hybrid molecules comprise for instance a cationic peptide with an antimicrobial action and/or a peptide fragment of ubiquicidine and/or a derivative thereof and one or more effector molecules.

The present invention relates to the new medical use of a per se knownpeptide, which will be referred to as “ubiquicidine” in thisapplication. The invention further relates to new peptide fragmentsderived from this peptide, optionally in modified form or provided witha (radioactive) label, and the use hereof in prophylaxis, therapy anddiagnostics of infections in humans and animals. The invention alsorelates to new antimicrobial and diagnostic agents on the basis of thepeptide, the peptide fragments and/or modified versions thereof,optionally in the form of combination preparations. Finally, theinvention also provides a new method for preparing radioactivelylabelled peptides with antimicrobial activity.

In an increasing number of cases the use of what are called “classic”antibiotics is not sufficient for the treatment of infectious diseases.Many bacteria strains have built up resistance against the known classesof antibiotic and in the last thirty years no new classes of antibiotichave been discovered. There are few or no adequate agents againstmycobacteria. And other micro-organisms, such as fungi, and determinedparasites are also sometimes difficult to treat with existingantimicrobial agents. In view of the above, a new class of antimicrobialagents is highly desirable.

At present, two new types of antimicrobial agents are attractingattention. On the one hand there are the carbohydrate-type agents. Inaddition, research is focussing on peptides, particularly (cationic)peptides, with antimicrobial activity. Cationic peptides contain arelatively large number of positively charged amino acids, such asarginine and lysine, and therefore carry a net positive charge, usuallyof at least +2, but often +4 or more. Antimicrobial peptides are animportant component of the natural defence of most living organismsagainst infections. Many such antimicrobial peptides are cationic. Inhumans and other mammals such peptides, such as the defensins, are animportant protein-like constituent of for instance neutrophilgranulocytes. These cells are already involved at a very early stage inthe defence against micro-organisms and in acute inflammation reactions.In addition, such peptides are also produced by many other cells,including epithelial cells, which are strategically located in relationto invading micro-organisms.

In the research which resulted in the present invention, it was foundthat the per se known peptide FAU S30 (which has now been called“ubiquicidine” by the present inventors) has antimicrobial action. Itwas further found that peptide (fragments) derived from this peptidealso have an antimicrobial action to a lesser or greater extent. Thesepeptide (fragments) as such have not been described previously and aretherefore still new.

On the basis of this conclusion, the present invention provides the useof ubiquicidine or optionally modified peptide (fragments) derivedtherefrom for preparing a drug for the treatment, diagnostics orprophylaxis of infections in humans and animals.

The advantage of ubiquicidine and fragments thereof is that they notonly have an antimicrobial and immunomodulating action, but that theyalso make their way in the body in targeted manner to the actual site ofinfection and accumulate there. These peptide (fragments) are thereforeinfection-seeking.

In this application “antimicrobial action” is understood to mean anyinhibiting or otherwise negative effect on bacteria, viruses, protozoa,parasites and fungi.

“Immunomodulating action” is understood in this application to mean anystimulating effect on body cells of humans and/or animals involved inthe defence against infections.

“Ubiquicidine” is understood in this application to mean a peptide of6.654 kD with an amino acid sequence as shown in FIG. 1.

Peptide fragments derived from ubiquicidine comprise a preferablycontinuous series of at least 3, preferably at least 8 amino acids fromthe amino acid sequence of ubiquicidine as shown in FIG. 1. For anaverage skilled person it is simple to ascertain whether a peptidefragment with a series of preferably continuous amino acids chosen fromthe amino acid sequence of ubiquicidine does actually have antimicrobialactivity and thus meets the requirements of the invention. A simplestandard test for determining antimicrobial activity is for instance theuniversally known growth-killing test, i.e. determining of theconcentration of an antimicrobial agent which kills 99% of themicro-organisms (IC 99%). Designated by “peptide (fragments)” in thisapplication are therefore all amino acid chains which are smaller thanthe ubiquicidine itself, but the amino acid sequence of which is to befound, preferably continuously, in the ubiquicidine. The length of suchpeptide (fragments) can vary from 3 to 58 amino acids, wherein possibleextra amino acids added as modification are not included.

Examples of peptide (fragments) are the peptides of which the sequenceis shown in FIG. 1. Ubiquicidine (18-35)-D-alanine has as extra additiona D-alanine at both ends. Of the peptide fragments shown in FIG. 1,ubiquicidine (1-18), ubiquicidine (18-35) and ubiquicidine (29-41) areparticularly recommended. In the above described test the activity ofthese fragments lies around 1 μM. This is a particularly goodantimicrobial activity. In principle however, all the above definedpeptides, which display some inhibiting action or other onmicro-organisms, fall within the invention. Peptides with an IC 99% of amaximum of 25 μM, preferably a maximum of 10 μM, most preferably amaximum of 1 μM are however recommended.

In order to modify their activity, for instance to further increase it,or to inhibit or prevent degradation by enzymes, particularlypeptidases, both the peptide (ubiquicidine) and the fragments can bemodified in different ways. Modification is any variation from thenaturally occurring amino acid chain. Modifications may be mutuallinking in reverse sequence of at least a part of the amino acids of thepeptide or a peptide fragment. When all amino acids of a peptide(fragment) are thus reversed, this is referred to as “reverse peptide(fragment)”.

One or more of the amino acids from the original peptide (fragment) canalso be replaced by a stereoisomer of that amino acid. The L-isomers ofamino acids occur in the body. The D-stereoisomers can be degraded muchless easily by enzymes present in the body and bacterial enzymes. Such amodification ensures that the peptide (fragment) in the body remainsintact longer and can exert its effect longer. A similar modificationconsists of extending the original amino acid chain at one or both endswith one or more groups protecting against degradation, such as D-aminoacids, for instance D-alanine.

All the amino acid chains modified in the above described manner orvarying in other manner from the corresponding native peptide (fragment)will be designated in this application with the term “derivative”. Thesecan be derivatives of the ubiquicidine as well as of fragments thereof.

The invention further relates to so-called “hybrid molecules”, whichcomprise a (cationic) peptide with an antimicrobial action and/or apeptide fragment and/or a derivative thereof according to the inventiontogether with one or more effector molecules. The effector molecule canassume different forms, such as an amino acid chain, which is capable ofbinding to a micro-organism and/or substances secreted bymicro-organisms or expressed on the surface thereof. An example of suchan effector molecule is an endotoxin-binding peptide.

Another type of effector molecule can consist of a virus protein. Such avirus protein/antimicrobial peptide can enter the host cell, in whichthe micro-organism for combatting is situated, in the known manner of avirus and the peptide can exert its antimicrobial action therein.

The effector molecule can further be a detectable label, such as aradionuclide, chosen from the group consisting of technetium 99m(Tc-99m), iodine 123(I-123) and 131 (I-131), bromine 75 (B-75) and76(B-76), lead 203 (Pb-203), gallium 67 (Ga-67) and 68 (Ga-68), arsenic72 (As-72), indium 111 (In-111), 113m (In-113m) and 114m (In-114m),ruthenium 97 (Ru-97), copper 62 (Cu-62), 64 (Cu-64) and 67 (Cu-67), iron52 (Fe-52), manganese 52m (Mn-52m), chromium 51 (Cr-51), rhenium 186(Re-186) and 188 (Re-188), terbium 161 (Tb-161) and yttrium 90 (Y-90).The radionuclide (also called “emitter”) can also fulfil a curativefunction. Paramagnetic labels, such as fluorine 19 (F-19), sodium 23(Na-23), phosphorus 31 (P-31), gadolinium 157 (Gd-157), manganese 55(Mn-55), dysprosium 162 (Dy-162), chromium 52 (Cr-52) and iron 56(Fe-56) can also be used.

According to the invention combinations of effector molecules canlikewise be linked to the peptide. An example thereof are a cell-bindingpeptide and an emitter, wherein the cell-binding peptide and theantimicrobial peptide provide targeting of the hybrid molecule to thesite of infection and the antimicrobial peptide and the emitter providefor treatment or diagnosis.

Hybrid molecules of this type which consist of an antimicrobial peptide,peptide fragment or derivative thereof and at least one effectormolecule have not been described previously. The “hybrid molecules”according to the invention are not therefore limited to the ubiquicidineas antimicrobial peptide, but generally comprise hybrid moleculescomprising a (cationic) peptide with antimicrobial activity and/orfragments and/or derivatives thereof. Examples of other suchantimicrobial peptides are α- and β-defensins, protegrins, serprocidins,magainins, PR-39, cecropins and others (Martin et al. (1995) J.Leukocyte Biol. 58:128-136).

The invention relates to the variants of the peptide or fragmentsthereof comprehensively described above. These variants as well as thepeptide and the fragments can also be designated collectively in thisapplication as “peptide (fragments)”.

The invention further relates to an antimicrobial agent comprising asactive component ubiquicidine and/or peptide fragments thereof,derivatives of one of both and/or hybrid molecules containing at leastubiquicidine or other antimicrobial cationic peptides and/or peptidefragments thereof and/or derivatives thereof for use in the diagnostics,prophylaxis, monitoring or therapy of infections.

The antimicrobial agent according to the invention can contain only theactive component or take the form of a pharmaceutical composition inwhich one or more other carriers, diluents and the like are present. Theagent and the composition can have different forms of administration,such as for instance tablet, pill, capsule, injection, infusion,suppository, powder, suspension, solution, spray, emulsion, ointment,aerosol, plaster or cream and can be used for oral, anal, nasal,vaginal, intramuscular, subcutaneous, intravenous, intraperitoneal orlocal (topical) administration or administration by means of a cathetervia natural or artificial body openings. Other very specific examples offorms of administration are toothpaste, tooth varnish and catheterscoated with the active compound. These latter have a prophylacticaction.

Compositions according to the invention can be prepared by combining(i.e. mixing, dissolving et cetera) of the active component(s) withpharmaceutically and pharmacologically acceptable excipients with aneutral character (such as aqueous or non-aqueous solvents, stabilizers,emulsifiers, detergents, additives)., and further, where necessary,colorants, aromatic substances and/or flavourings. The concentration ofthe active component(s) in a pharmaceutical composition can vary between0.001% and 100% (w/v), depending on the nature of the treatment and themanner of administering. The dose for administering likewise depends onthe manner of administration and nature of the treatment. For the mousefor instance a dose of 1 to 10 μg/kg, for instance 4 μg/kg body weight,is suitable. The compositions according to the invention are suitablefor treatment of both humans and animals.

The invention further relates to the ubiquicidine, to peptide fragmentsthereof, to derivatives of one of both and to hybrid moleculescontaining at least ubiquicidine or other antimicrobial cationicpeptides, and/or peptide fragments thereof and/or derivatives thereoffor use in diagnostics, prophylaxis, therapy or monitoring ofinfections.

Infections, which can be treated with the agent are for instancedisorders caused by pathogenic Gram-positive (Staphylococcus aureus,Listeria monocytogenes including antibiotic-resistant strains of S.aureus (also called Multidrug-Resistant S. aureus (MRSA))), andGram-negative ((antibiotic-resistant) Klebsiella pneumoniae, Escherichiacoli, enterococci and Salmonella typhimurium) bacteria, micro-organismsdifficult to treat such as Mycobacterium avium and Mycobacteriumfortuitum, fungi such as Candida albicans, Cryptococcus neoformans andAspergillus fumigatis, viruses, in particular enveloped viruses, andparasites, such as Trypanosoma cruzi and Toxoplasma gondii. The use ofthe agent is however not limited to the infections stated here.

Because the peptide (fragment) according to the invention isinfection-seeking, it can be applied very well in the diagnostics ofinfections and pathology related thereto. If provided with a detectablelabel, for instance a radioactive label such as technetium 99m, it ispossible for instance by means of scintigraphy to determine some timeafter administering where in the body the peptide (fragment) issituated. This will also be the site where the infection for treatmentis situated. Such a labelled peptide (fragment) therefore has a dualpurpose. Not only is demonstrated where the infection is situated, butthe peptide (fragment) will also exert an antibiotic action due to itspresence at the site and thus reduce the infection. In this manner theeffect of the treatment can also be followed by looking at thelocalization of the peptide in time. This is called “monitoring”.

Each of the above mentioned radionuclides can in principle be used.Particularly recommended however is technetium 99m (^(99m)Tc). Thephysical half-life of this radionuclide amounts to 6 hours and, togetherwith the fact that particularly gamma radiation is emitted, this means alow radiation load for the patient. The relatively short half-lifemoreover has the clinical advantage that the examination can be repeatedrapidly. In addition, this radionuclide is readily obtainable via thecommercially available Mo-Tc-nuclide-generator.

It is found that peptide (fragments) labelled with technetium 99m canalready be detected after 15 minutes at the site of the infection. Theaccumulation of for instance gallium 67 takes at least 24 hours. Owingto the very rapid localization of peptide (fragments) labelled withtechnetium 99m, a rapid diagnosis is possible. Furthermore, technetium99m is mainly a γ emitter with a very small quantity of the much moreharmful β radiation, so there is a relatively low radiation load for thepatient. A more frequent administration is hereby possible. In addition,it has also been found that labelling r with technetium 99m has noadverse influence on the action of the peptide (fragment). In laboratoryanimal experiments no adverse effects or changed externalcharacteristics due to ^(99m)Tc-labelled peptide (fragments) have beenfound up to the present. In vitro studies have moreover shown that veryhigh concentrations of the antimicrobial peptide (fragments) are nottoxic for human body cells. Particular recommended therefore accordingto the invention as hybrid molecules are technetium 99m-labelledcationic peptides and fragments or derivatives thereof.

The peptide (fragments) according to the present invention demonstratethe infection itself and thus the location where the micro-organism issituated in the body. Known image-forming methods for detectinginfections, such as X-ray, echography and the like-are aimed atdemonstrating morphological changes which are the result of aninfection. It is very well possible however for the infection itself tohave already disappeared, while the morphological change still exists.In that case the treatment of the infection with for instanceantibiotics is simply continued while this is in fact no longernecessary. It is recommended in principle to cause a treatment with adetermined antimicrobial agent to be as short as possible in respect ofthe occurrence of resistances or allergies as a result of the agent. Thepeptide (fragments) according to the invention are infection-seeking andtherefore make their way to the site of the infection itself and canalso be made visible there. As soon as the infection has disappeared,this is shown by the fact that the peptide (fragment) no longeraccumulates at the site of the (former) infection. The treatment canthen be stopped. Using labelled peptides, infections can also bedistinguished from inflammation processes. Infections occur when thebody reacts to the presence of a foreign living organism. “Inflammation”is a general name for reactions of the body to foreign stimuli, such asparticles, molecules, but also live bacteria. The peptide only reacts inthe case of infections.

The invention further relates to combination preparations which, inaddition to ubiquicidine and/or a peptide fragment thereof and/or aderivative thereof and/or a hybrid molecule, contain one or more otheractive components. Combinations with “classic” antibiotics or withantiviral or antifungal agents can for instance be envisaged.

The invention further relates to a method for labelling antimicrobialpeptides, particularly cationic peptides, more particularly ubiquicidineand peptides derived therefrom and defensins. Such a method comprises ofplacing the peptide for labelling in contact with a tin(II) salt, aborohydride and a radioactive label in the presence of alkali, asdescribed in Pauwels et al. (Nucl. Med. Biol. 20, 825-833 (1993)), butwherein the peptide is modified with MAG3 (mercapto-acetylglycine-glycine-glycine). Prior to labelling the modified peptide isheld at about 100° C. for 10 minutes. Particularly in the case of smallpeptides or peptides carrying no sulphur groups, the MAG3 modificationresults in considerably higher labelling efficiencies.

The whole is stirred at a suitable temperature for a determined time,for instance 1 to 60 minutes, preferably 5 to 30 minutes. Thetemperature depends on the temperature sensitivity of the peptide, butwill usually lie between room temperature and 40° C., and willpreferably be about 37° C.

The tin(II) salt is preferably tin(II)pyrophosphate. The borohydride ispreferably sodium borohydride or potassium borohydride. The tin(II) saltand the borohydride are advantageously used in a ratio between 1:1 and1:10, preferably 1:4 in quantities of respectively 0.5-5 μl and 2-10 μl.In preference 0.1 M sodium hydroxide is used as alkali.

The radioactive label is advantageously ^(99m)Tc-pertechnetate, but¹⁸⁶Re-perrhenate can also be used. Standard solutions of suchradioactive labels are commercially available. In the method accordingto the invention 0.05-0.5 ml, preferably 0.1 ml of such a solution isused.

A particularly advantageous manner of preparing ubiquicidine, andoptionally the fragments, derivatives and hybrid molecules, is by meansof transgenic animals. For this purpose the method comprises oftransforming an animal egg-cell with a gene construct which codes forthe ubiquicidine, peptide fragment, derivative or hybrid molecule,regenerating a transgenic animal from the transformed egg-cell andisolating the ubiquicidine, peptide fragment, derivative or hybridmolecule from a tissue or bodily fluid of the animal, for instance milk.The products can of course also be synthesized.

The present invention will further be elucidated on the basis of theaccompanying examples, which are only given by way of illustration butdo not limit the invention. Reference is made in the examples to thefollowing figures, in which:

FIG. 1 shows the amino acid sequence of ubiquicidine and derivedpeptides

FIG. 2 shows the antimicrobial effect of ubiquicidine in respect ofKlebsiella pneumoniae and Staphylococcus aureus

FIG. 3 shows the effect of ubiquicidine (18-35) on herpes simplex virusinfection of Vero cells

FIG. 4 shows the effect of ubiquicidine (18-35) on Mycobacteriumfortuitum

FIG. 5 shows the effect of ubiquicidine (18-35) and ubiquicidine (18-29)on (antibiotic-resistant) Staphylococcus aureus

FIG. 6 shows the effect of ubiquicidine (18-35) and D-alanine-protectedubiquicidine (18-35) on Klebsiella pneumoniae

FIG. 7 shows the speed of ubiquicidine (18-35) and D-alanine-protectedubiquicidine (18-35) with which Staphylococcus aureus is eliminated

FIG. 8 shows the effect of ubiquicidine (18-35) and D-alanine-protectedubiquicidine (18-35) on (antibiotic-resistant) Staphylococcus aureus

FIG. 9 shows the effect of D-alanine-protected ubiquicidine (18-35) on(antibiotic-resistant) Escherichia coli

FIG. 10 is a scintigram of intraperitoneally administered^(99m)technetium-labelled ubiquicidine (18-35) in a mouse infected withStaphylococcus aureus

FIG. 11 is a schematic view of the experimental infection and treatmentof mice

FIG. 12 shows the accumulation of ^(99m)technetium-labelled ubiquicidine(18-35), ubiquicidine (1-18), defensins and human IgG in the thighmuscle infected with Klebsiella pneumoninae

FIG. 13 shows the accumulation of 99mTc-labelled ubiquicidine 18-35 in anidus but not in inflammations

FIG. 14 shows the effect of ubiquicidine (18-35), ubiquicidine (1-18)and defensins on an experimental infection with Staphylococcus aureusand Escherichia coli

FIG. 15 shows the antimicrobial effect of ubiquicidine 29-41 and 18-35and defensin-1 in mice.

EXAMPLES Example 1

Antimicrobial Action of Ubiquicidine

1. Introduction

By means of gel filtration and reverse phase HPLC a peptide was isolatedfrom the cytosol fraction of murine RAW 264.7 macrophages activated withinterferon γ and cells of the human H292 bronchial epithelial cell linestimulated in different ways. The latter could be stimulated withbacterial products (endotoxin, lipoteichoinic acid), phorbol ester, andbronchial pathogens (Haemophilus influenzae, Streptococcus pneumoniaeand para-influenza virus 3). The isolated peptide was calledubiquicidine.

2. Materials and Method

2.1. Isolation of Ubiquicidine

The method of isolating ubiquicidine from cytosol fractions of cells hasbeen previously described for the isolation of antimicrobial proteinsfrom cell lysates and cell membrane fractions (Hiemstra et al. (1993)Infect Immun. 61:3038-3046). The cells were cultured in RPMI 1640 mediumwith antibiotics and 10% heat-inactivated foetal calf serum. The cellswere subsequently harvested, washed and resuspended in 10 mM sodiumphosphate buffer (pH 7.4) enriched with a cocktail of proteaseinhibitors:

Using nitrogen cavitation, a cell lysate was obtained whereafter bymeans of ultracentrifugation at 27,000×g a membrane fraction and acytosol fraction were obtained. The proteins in the cytosol fractionwere extracted using 5% acetic acid and the acid extract was dialyzedand subsequently placed on a P60 column.

The fractions originating from this column were tested for antimicrobialactivity. The ubiquicidine-containing fractions were pooled and furtherseparated by means of HPLC on a C18 column with heptafluorobutyric acidas “ion pairing molecule” in the eluent. The HPLC fractions werelikewise tested for antimicrobial activity and immunoreactivity using anantiserum against the N-terminal part of the ubiquicidine. The pooledfractions contain pure ubiquicidine.

2.2. Biochemical Characterization

The sequence of the N-terminal amino acids of purified ubiquicidine wasdetermined by means of automated Edman degradation and a peptidesequencer 477A equipped with a PTH amino acid analyzer 120A (AppliedBiosystems, Foster City, Calif.). The sequence results were subsequentlyanalysed using the GeneWorks software package (Intelligenetics, MountainView, Calif.). Molecular weight of ubiquicidine was determined usingmass spectrometry (laser desorption time-of-flight mass spectrometry;Lasermat, Finnigan MAT LTD, Hemel Hempstead, UK). For the immunologicalidentification of ubiquicidine use was made of a rabbit antiserumspecific to the N-terminal part of ubiquicidine (ubiquicidine 1-18) andWestern blotting.

2.3. Tests for Antimicrobial Activity In Vitro

Different techniques were used to test for the antimicrobial activity ofubiquicidine and peptides derived therefrom. The gel overlay assay andthe radial diffusion assay have been previously described (Hiemstra etal. (Infect. Immun. 63, 3038-3046 (1993)). In the growth-killing curvedetermination which was used to investigate the IC 99% of the peptide,(mid-log or stationary phase) bacteria (Klebsiella pneumoniae (A) andStaphylococcus aureus (B) were exposed for 60 minutes at 37° C. toincreasing concentrations of the ubiquicidine, whereafter the number ofliving bacteria in the suspension was determined using microbiologicalplate techniques (Colony Forming Units, CFU). As negative controls,bacteria were exposed to peptide 4 (a synthetic peptide derived from HIVglycoprotein 120), ubiquicidine (18-29) or no peptide.

The results of such experiments are shown in CFUs in FIG. 2.

3. Result

The ubiquicidine is a 6.7 kD ribosomal cationic peptide with a pI of12.67. From sequence determination of the 18 N-terminal amino acids ofthe isolated peptide, it was found that these corresponded wholly withthe N-terminal part of the S30 part of the expression product of theFinkel-Biskis-Reilly murine sarcoma associated ubiquitously expressed(FAU) gene which occurs inter alia in humans and mice. The molecularweight of the FAU S30 and the ubiquicidine were also found tocorrespond. It is therefore assumed that it is the same peptide.

From the determination of the in vitro antimicrobial action ofubiquicidine it was found that the ubiquicidine can kill micro-organismsvery rapidly (<10 minutes) and effectively (3-4 log reduction). FIG. 2shows mid-log Klebsiella pneumoniae (A) and Staphylococcus aureaus (B),which were exposed for 60 minutes at 37° C. to increasing concentrationsof purified ubiquicidine in 10 mM sodium phosphate buffer. In thecontrol incubations the bacteria multiplied a number of times (notshown). The minimal inhibiting concentration for said micro-organismswas found to lie between 0.08 and 0.16 μM, 1.5 μM ubiquicidineeliminates Klebsiella pneumoniae (A) almost completely, while thereduction in the number of Staphylococcus aureus (B) amounts to 2 log.

Example 2

Antimicrobial Action of Peptide Fragments

1. Introduction

A number of peptide fragments were derived from the native ubiquicidineand the antimicrobial activity thereof was determined.

2. Materials and Methods

2.1. Production of Synthetic Peptides

Peptides were prepared using an Abimed AMS multiple peptide synthesizerand a fixed phase (tentagel AC, a polymer of polyethylene glycol spacerlinked to a polystyrene matrix) (de Koster et al. (1995) J. Immunol.Methods 187:179-188). After completion of the synthesis the peptide wasreleased from the fixed phase using a trifluoroacetic acid water(19:1)mixture and the peptides were subsequently precipitated with an etherpentane(1:1) mixture at 20° C. After centrifugation the obtainedpep-tides were dried at 40° C. for 15 minutes. The peptides weresubsequently dissolved in 10% acetic acid and concentrated by means ofvacuum centrifugation. The purity of the peptides was determined usingHPLC. An overview of the synthesized peptides derived from ubiquicidineis given in FIG. 1. The antimicrobial activity of these peptidefragments was determined as described in Example 1 under 2.3.

2.2. Antimicrobial Effect on Herpes Simplex Virus (HSV)

HSV was incubated for 60 minutes with increasing concentrations of thepeptide fragment ubiquicidine (18-35) at 37° C. The virus preparationwas subsequently added to Vero cells in diverse dilutions. After 3 daysat 37° C. the cytopathogenic effect of the virus on Vero cells wasdetermined, with finally made it possible for the virus titre to becalculated. FIG. 3 shows the result.

2.3. Antimicrobial Effect on Mycobacterium Fortuitum

About 10⁶ Mycobacterium fortuitum were incubated for different intervalsat 37° C. with 14 μM or 52 μM ubiquicidine (18-35) and the number ofliving mycobacteria in the suspensions was then determined usingmicrobiological techniques. The result is shown in FIG. 4.

2.4. Antimicrobial Effect on Staphylococcus Aureus

About 10⁶ bacteria of multidrug resistant Staphylococcus aureus (MRSA)and antibiotic-sensitive S. aureus were exposed for 60 minutes at 37° C.to different concentrations of ubiquicidine (18-35), whereafter thenumber of living bacteria in the suspensions was determinedmicrobiologically. As negative control high concentrations ofubiquicidine (18-29), peptide 4 and no peptide were used. The result isshown in FIG. 5.

3. Results

Research into the effect of the different peptides on Klebsiellapneumoniae and Staphylococcus aureus demonstrated antimicrobial activityof ubiquicidine (1-18), ubiquicidine (18-35) and ubiquicidine (29-41).The other peptides were found to be considerably less potent orinactive.

FIG. 3 shows the results of the experiment with HSV. This shows that anincreasing concentration of peptide results in a decrease in the virustitre.

FIG. 4 shows that ubiquicidine (18-35) kills M. fortuitum for a periodof 3 hours, whereafter the peptide then shows a bacteriostatic effect.Repeated administration at 3 and 7 hours after the first dose results inpractically complete elimination of the mycobacteria. In the controlincubations M. fortuitum was found to proliferate. In additional controlexperiments no indication was found for agglomeration of themycobacteria due to ubiquicidine (18-35) (not shown).

FIG. 5 shows that the peptide fragment ubiquicidine (18-35) results in amarked decrease in the number of CFUs of different Staphylococcus aureusstrains.

Example 3

Modified Peptide Fragments and their Activity

1. Introduction

A number of the peptide (fragments) described in Example 2 was furthermodified in different ways by adding an extra D-alanine at the beginningand/or end as protection against exopeptidase activity. Theantimicrobial activity of several “derivatives” obtained in this mannerwas likewise determined.

2. Materials and Methods

2.1. Production Of Modified Peptides

D-alanine-protected peptides were prepared as described above (Example2, ad 2.1) in this application.

2.2. Antimicrobial Effect on Staphylococcus Aureus

Staphylococcus aureus (5×10⁵ bacteria) was exposed for different periodsat 37° C. to 7 μM ubiquicidine (18-35) and D-alanine-protectedubiquicidine (18-35), whereafter the number of living bacteria in thesuspension was quantified microbiologically.

In addition, different strains of (multidrug resistant) Staphylococcusaureus were incubated for 60 minutes at 37° C. with increasingconcentrations of D-alanine-protected and unprotected ubiquicidine(18-35), whereafter the number of living bacteria in the differentsuspensions was determined microbiologically.

2.3. Antimicrobial Effect on Klebsiella Pneumoniae

About 5×10⁶ Klebsiella pneumoniae were exposed for 60 minutes at 37° C.to increasing concentrations of ubiquicidine (18-35) andD-alanine-protected ubiquicidine (18-35) and the number of live bacteriawas subsequently measured microbiologically.

2.4. Antimicrobial Effect on Escherichia Coli

About 10⁶ antibiotic-resistant Escherichia coli and antibiotic-sensitiveE. coli (parent strain of the resistant bacteria) were exposed for 60minutes at 37° C. to increasing concentrations of D-alanine-protectedubiquicidine (18-35), whereafter the number of living bacteria wasdetermined microbiologically.

3. Results

Comparison of antimicrobial activities of the D-alanine-protected andthe unprotected ubiquicidine (18-35) in respect of Klebsiella pneumoniaein vitro showed that the D-alanine-protected variant is much more potentin eliminating the bacteria than the unprotected ubiquicidine (18-35)peptide (FIG. 6).

The maximum killing effect by both variants of ubiquicidine(ubiquicidine (18-35) and D-alanine-protected ubiquicidine (18-35)) onStaphylococcus aureus was achieved within 15 minutes (FIG. 7). The speedof elimination of Staphylococcus aureus bacteria by the two types ofubiquicidine peptide is identical (FIG. 7).

The results further demonstrated that the D-alanine-protectedubiquicidine kills (multidrug resistant) Staphylococcus aureus moreeffectively than the unprotected variant (FIG. 8).

Very surprising is the observation that the D-alanine-protectedubiquicidine (18-35) can kill antibiotic-resistant Escherichia coli muchmore effectively than the antibiotic-sensitive parent strain ofEscherichia coli (FIG. 9). 1 μM D-alanine-protected ubiquicidine reducesthe number of antibiotic-resistant bacteria to below the detectionlimit. A comparable antimicrobial effect relative to the parent strainis only achieved with 14 μM of the peptide. This data shows thatantibiotic-resistant bacteria can be eliminated very effectively bypeptides derived from ubiquicidine.

Example 4

Peptide Fragments Labelled with Technetium 99m

1. Introduction

A hybrid molecule was prepared by labelling the peptide fragments withthe emitter ^(99m)Tc. This example illustrates the manner of labellingaccording to the invention.

2. Materials and Method

Labelling of peptide D (ubiquicidine (18-35)), and theD-alanine-protected ubiquicidine (18-35) with ^(99m)Tc was performedusing a method according to the invention. For this purpose 10 μl of aMAG3-derived peptide solution (2 mg/ml in 0.01 M sodium phosphate pH3.0) was added to 2 μl of a tin(II)pyrophosphate solution (0.5 mg/ml).Immediately thereafter 4 μl of a 10, mg/ml KBH₄ solution (Sigma ChemicalCompany, St. Louis, Mo., US) in 0.1 M NaOH was added. After adding 0.1ml of a ^(99m)Tc-sodium pertechnetate solution (20 MBq, MallinckrodtMedical B.V., Petten, Netherlands) the mixture was stirred at roomtemperature for 30 minutes.

The radiochemical purity of peptides labelled with ^(99m)Tc wasdetermined after precipitation with 20% trichloroacetic acid (TCA),instantaneous thin-layer chromatography (ITLC) and HPLC. Summarizing,this took place by analysing 20 μl of a freshly prepared^(99m)Tc-defensin-1 or ^(99m)Tc-IgG on a superose 12 column (Pharmacia,Upsala, Sweden), linked to an LKB Bromma HPLC 2249 chromatography pump(LKB, Upsala, Sweden) and an on-line NAI (Tl)-crystal-gamma-detectionsystem (Raytest Steffi, Germany). The buffer which was used foranalysing the ^(99m)Tc-labelled compounds was 14 mM sodiumphosphate-buffered salt solution (PBS) pH 7.5 with a flow rate of 1ml/minute. Labelling yields of ⁹⁹Tc-labelled peptides were determinedafter precipitation with 209 TCA, HPLC analysis and ITLC analysis andwere respectively more than 90%, more than 90% and more than 95%.

Example 5

Accumulation of the Labelled Peptide at the Site of Infection

1. Introduction

In order to demonstrate that the peptide (fragment) according to theinvention is infection-seeking, the localization of ⁹⁹Tc-labelledpeptides (ubiquicidine (18-35), ubiquicidine (1-18) and defensins inaddition to IgG as control) was determined using a γ-camera.

2. Materials and Method

Mice were infected intramuscularly with about 10⁶ Staphylococcus aureusbacteria (ATCC 25923) and subsequently injected intraperitoneally with25 μg ^(99m)Tc-peptide. Mice were also injected intramuscularly withabout 1×10⁸ heat-killed (1 hour, 100° C.)S. aureus, 1 μg endotoxin or100 ng phorbol myristate acetate (PMA) in order to cause sterileinflammations. At different points in time after injection of thepeptide the radioactivity was measured in the circulation (heart),determined organs (liver, kidney, bladder and spleen) and in both thighmuscles using a γ-camera. Accumulation of the labelled peptide at thesite of infection in the right thigh muscle is shown in FIG. 10.

3. Results

The results showed a very short half-life of the peptides in thecirculation, i.e. t_(half)<15 minutes. The largest part of the injectedlabelled peptides (>60%) is removed via the liver, kidneys and bladder,but a part of the peptides (1-2% of the injected dose) arrives at thesite of infection in the thigh muscle (FIG. 10).

Example 6

Antimicrobial Activity In Vivo

1. Introduction

The accumulation and antimicrobial activity in vivo of a number ofubiquicidine fragments was determined.

2. Materials and Methods

2.1. Infection Model

Reference is made to FIG. 11 for a schematic view of the experimentalinfection and treatment of the mice. In summary, mice-were infectedintramuscularly in the right thigh muscle with about 10⁶ bacteria andafter 5 minutes injected intraperitoneally with about 25 μg (labelled)peptide. At different points in time after injection of the peptide theanimals were killed and the right thigh muscle was removed, homogenized,and finally the number of bacteria in the homogenate was determinedusing microbiological plate techniques, or accumulation of the labelledpeptide was determined by means of a γ-camera. This test involvedanimals which were normal and immunocompromised (injection withcyclophosphamide, “total body” radiation).

2.2. Infection-seeking Effect of Peptides According to the Invention

Mice were infected in the right thigh muscle with Klebsiella pneumoniaeand 25 μg ^(99m)Tc-labelled ubiquicidine (1-18) or ubiquicidine (18-35)was subsequently injected intraperitoneally. At different points in timeafter injection of the peptide the amount of activity in the right(test) and left (control) thigh muscle of the mouse was measured using aγ-camera. The results are shown in FIG. 12 as a ratio of the values inthe right thigh muscle and the left thigh muscle, i.e. “target tonon-target ratio”. For the purpose of comparison the results for humandefensin and IgG are also shown. The target to non-target ratios forinfections and sterile inflammations were also compared (FIG. 13).

2.3. Effect of Antimicrobial Peptides on Experimental Infections

Mice were infected in the right thigh muscle with Klebsiella pneumoniae(A) or Staphylococcus aureus (B). 5 minutes later, 25 μg ubiquicidine(18-35) or ubiquicidine (1-18) was injected intraperitoneally. 24 hoursafter administering of the peptide the animals were killed and thenumber of bacteria in the right thigh muscle was quantifiedmicrobiologically. As positive control, animals were injectedintraperitoneally with human defensin and as negative control with thesolvent of the antimicrobial peptides. The result is shown in FIG. 14.The mice were also injected with 150 mg cyclophosphamide/kg body weight.Four days afterwards the animals were infected in the right thigh musclewith K. pneumonia and a day later different quantities of ubiquicidine(18-35), ubiquicidine (29-41) or defensin-1 were injected intravenously.Twenty-four hours after administering of the peptide the animals werekilled and the number of bacteria in the right thigh muscle wasquantified microbiologically. As control, normal animals were treated inidentical manner. The result is shown in FIG. 15.

3. Results

The accumulation of the tested peptides was found to be maximal 4 hoursafter administration and subsequently decreases in the course of time(FIG. 12). It is notable that the maximum accumulation of⁹⁹Tc-ubiquicidine (1-18) and ⁹⁹Tc-ubiquicidine (18-35) is reached muchsooner than ⁹⁹Tc-IgG. This observation implies that ⁹⁹Tc-ubiquicidinepeptides can be of importance for faster diagnostics of infections.Comparable results were found when the ⁹⁹Tc-peptide was administeredintravenously 24 hours after infection.

The above stated pharmacological data shows that ubiquicidine (1-18) andubiquicidine (18-35) accumulate rapidly in the infected thigh muscle.The results of our experiments into the effect of these peptides on thenumber of bacteria in the muscle demonstrate that ubiquicidine (18-35)eliminates bacteria more effectively than ubiquicidine (1-18) anddefensins (FIG. 12). These in vivo results correspond very well with theresults of the in vitro experiments.

FIG. 14 shows that particularly ubiquicidine (18-35) also has a markedbactericidal effect in vivo which is better than that of defensin.

The result in immunocompromised animals (FIG. 15) shows that thebactericidal effect in vivo is determined by a direct bactericidaleffect as well as by an immuno-modulating effect.

1. Peptide fragment derived from ubiquicidine having antimicrobialactivity and comprising a continuous series of between 6 to 18 aminoacids from the amino acid sequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPN ANS, (SEQ IDNO: 1) which does not include peptides having the amino acid sequenceKVHGSLARAGKVRGQTPKVAKQ (SEQ ID NO: 10) or AGKVRGQTPKVAKQEKKKKKT (SEQ IDNO: 11).
 2. Peptide fragment as claimed in claim 1, comprising acontinuous series of between 6 to 18 amino acids from the amino acidsequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPN ANS, (SEQ IDNO: 1) which does not include peptides having the amino acid sequenceKVHGSLARAGKVRGQTPKVAKQ (SEQ ID NO: 10) or AGKVRGQTPKVAKQEKKKKKT (SEQ IDNO: 11).
 3. Peptide fragment as claimed in claim 1, consisting of thefollowing amino acid sequences: ubiquicidine (1-18) KVHGSLARAGKVRGQTPK(SEQ ID NO: 2) ubiquicidine (29-41) TGRAKRRMQYNRR (SEQ ID NO: 3)ubiquicidine (18-29) KVAKQEKKKKKT (SEQ ID NO: 4) ubiquicidine (18-35)KVAKQEKKKKKTGRAKRR (SEQ ID NO: 5) ubiquicidine (29-35) TGRAKRR (SEQ IDNO: 7) ubiquicidine (42-59) FVNVVPTFGKKKGPNANS (SEQ ID NO: 8)ubiquicidine (36-41) MQYNRR (SEQ ID NO: 9).


4. Derivative of ubiquicidine or of a peptide fragment derived fromubiquicidine having antimicrobial activity and comprising a continuousseries of between 6 to 18 amino acids from the amino acid sequence ofubiquicidine: KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPNANS, (SEQ ID NO: 1) which derivative has an amino acid sequence which isat least partly the reverse of the amino acid sequence of thecorresponding original peptide.
 5. Derivative of a ubiquicidine or of apeptide fragment derived from ubiquicidine having antimicrobial activityand comprising a continuous series of between 6 to 18 amino acids fromthe amino acid sequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPNA NS, (SEQ IDNO: 1) wherein at least one of the amino acids from the original peptideis replaced by a stereoisomer of that amino acid.
 6. Derivative ofubiquicidine or of a peptide fragment derived from ubiquicidine havingantimicrobial activity and comprising a continuous series of between 6to 18 amino acids from the amino acid sequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPNA NS, (SEQ IDNO: 1) wherein the original amino acid chain is extended at one or bothends thereof with one or more groups, such as D-amino acids, protectingagainst degradation.
 7. Derivative as claimed in claim 6, wherein theoriginal amino acid chain is extended at one or both ends thereof withone or more groups of D-alanine.
 8. A method for the therapy of aninfection in humans and animals, comprising: a) administering anantimicrobial compound having antimicrobial activity selected from thegroup consisting of ubiquicidine, a derivative of ubiquicidine, and apeptide fragment derived from ubiquicidine and comprising a continuousseries of between 6 to 18 amino acids from the amino acid sequence ofubiquicidine: KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPNANS (SEQ ID NO: 1); and b) treating the infection, wherein theantimicrobial action of the compound results in inhibiting or otherwiseexerting a negative effect on the infection.
 9. The method of claim 8,wherein the peptide fragment comprises a peptide fragment, comprising acontinuous series of between 6 to 18 amino acids from the amino acidsequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPN ANS, (SEQ IDNO: 1) with the exception of peptides having the amino acid sequenceKVHGSLARAGKVRGQTPKVAKQ (SEQ ID NO: 10) or AGKVRGQTPKVAKQEKKKKKT (SEQ IDNO: 11).
 10. The method of claim 8, wherein the derivative comprises aderivative of ubiquicidine or of a peptide fragment derived fromubiquicidine and comprising a continuous series of between 6 to 18 aminoacids from the amino acid sequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPN ANS, (SEQ IDNO: 1) which derivative has an amino acid sequence which is at leastpartly the reverse of the amino acid sequence of the correspondingoriginal peptide.
 11. The method of claim 8, wherein the microbialinfection is caused by a microorganism selected from the groupconsisting of Gram-positive bacteria, Gram-negative bacteria, fungi andviruses.
 12. Antimicrobial agent, comprising at least a suitablequantity of one or more active components chosen from ubiquicidineand/or peptide fragments derived from ubiquicidine, said ubiquicidineand/or said peptide fragments derived from ubiquicidine havingantimicrobial activity, and comprising a continuous series of between 6to 18 amino acids from the amino acid sequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPN ANS (SEQ ID NO:1).
 13. The method of claim 8, wherein the compound comprises anantimicrobial agent, comprising at least a suitable quantity of one ormore active components chosen from ubiquicidine, peptide fragmentsderived from ubiquicidine and comprising a continuous series of between6 to 18 amino acids from the amino acid sequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPN ANS (SEQ ID NO:1).
 14. Method for monitoring a treatment, comprising: a) administeringan antimicrobial agent having antimicrobial activity, comprising atleast a suitable quantity of one or more active components chosen fromubiquicidine, peptide fragments derived from ubiquicidine and comprisinga continuous series of between 6 to 18 amino acids from the amino acidsequence of ubiquicidine:KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVP TFGKKKGPNANS (SEQ ID NO:1), b) observing the localization of the agent in time, and c) followingthe effect of the treatment, wherein the antimicrobial action of thecompound results in inhibiting or otherwise exerting a negative effecton the infection.
 15. Antimicrobial agent as claimed in claim 12,further comprising at least one excipient.
 16. Method for providingprophylactic action against an infection in humans or animals,comprising: a) administering a peptide fragment, derived fromubiquicidine having antimicrobial activity and comprising a continuousseries of between 6 to 18 amino acids from the amino acid sequence ofubiquicidine: KVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNRRFVNVVPTFGKKKGPNANS, (SEQ ID NO: 1) which does not include peptides having theamino acid sequence KVHGSLARAGKVRGQTPKVAKQ (SEQ ID NO: 10) orAGKVRGQTPKVAKQEKKKKKT (SEQ ID NO: 11) in the form of a coating, whereinsaid administration results in preventing the infection.